Sustained-release liposomal anesthetic compositions

ABSTRACT

The invention provides a method for obtaining local anesthetics encapsulated in liposomes, such as multi vesicular liposomes, with high encapsulation efficiency and slow release in vivo. When the encapsulated anesthetic is administered as a single intracutaneous dose, the duration of anesthesia and half-life of the drug at the local injection site is increased as compared to injection of unencapsulated anesthetic. The maximum tolerated dose of the encapsulated anesthetic is also markedly increased in the liposomal formulation over injection of unencapsulated anesthetic. These results show that the liposomal formulation of local anesthetic is useful for sustained local infiltration and nerve block anesthesia.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/097,756, filed Apr. 1, 2005, which is a continuation of InternationalApplication No. PCT/US98/19583 filed Sep. 18, 1998, which claimspriority under 35 U.S.C. §119(e) to U.S. Provisional Application No.60/059,233, filed Sep. 18, 1997, all of which are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to liposomal formulations of compounds such asdrugs. More particularly this invention relates to methods ofencapsulating anesthetics in multivesicular liposomes with highefficiency and sustained in vivo rates of release.

2. Description of the Related Art

A local anesthetic's duration of action following administration isusually sufficiently long to cover the pain inflicted during mostsurgical procedures. However, the duration of action is not long enoughto cover most post-operative pain, or pain from many invasive diagnosticprocedures, or from injuries. Continuous infusion or repeatedinfiltration of a local anesthetic into a surgical wound, diagnostic“port” or injury site is impractical. Therefore, a sustained-releaseformulation of a local anesthetic would be useful for pain management,especially in view of the current trend for out-patient surgeries andemergency care centers. Desirably, such formulations are useful intrauma and diagnostic pain, as well.

Several approaches to develop sustained-release formulations of localanesthetics have been described in the literature. For example,polylactic-co-glycolic acid polymer micro spheres containing bothbupivacaine and dexamethasone have produced long duration of localanesthesia. Crystalline forms of local anesthetics have also been shownto have long duration of action. Lipophilic bupivacaine free-baseincorporated into the membranes of multilamellar liposomes andproton-gradient-loaded large unilamellar liposomes have shown efficacylasting 6 to 11 hours.

Multivesicular liposomes (MVL) are being developed as a lipid-basedsustained-release drug delivery system for local, regional or systemicdrug delivery. Sustained release of many water-stable drugs encapsulatedinto MVL has been shown in animal models via intrathecal, subcutaneous,intraperitoneal and epidural routes of administration, as well as inhuman patients via intracerebroventricular, intrathecal, subcutaneousand epidural routes. A multicenter, randomized phase III clinical trialof a MVL formulation of cytotoxic agent cytarabine has shown that thisformulation is more efficacious than free cytarabine in treatingleptomengial carcinoma.

MVL are defined as liposomes containing multiple non-concentric chamberswithin each liposome particle, resembling a “foam-like” matrix. Suchparticles are to be distinguished from multilamellar vesicles (MLV),also known as a multilamellar liposome, which contain multipleconcentric chambers within each liposome particle. A further distinctparticle is the unilamellar vesicle (ULV), also known as a unilamellarliposome, which encloses a single internal aqueous compartment. Thepresent invention relates to MVL. The prior art describes thepreparation of MVL (Kim et al., Biochim. Biophys. Acta 728, 339-348,1983).

Many of the cationic biologically active substances used in MVLencapsulation techniques are used as salts of monoprotic mineral acids(for example, as hydrochloride salts). The prior art has used suchcommonly available monoprotic mineral acid salts of cationicbiologically active substances for encapsulation into liposomes withoutany modification into a salt of diprotic or triprotic mineral acid. Theprior art has also used organic acids such as citric or glutamic acidsto effect encapsulation.

SUMMARY OF THE INVENTION

The invention provides local anesthetics encapsulated in multivesicularliposomes (MVL), that is, lipid vesicles having multiple non-concentricinternal aqueous chambers having internal membranes distributed as anetwork throughout the MVL. The chambers contain acids which areeffective to enable the encapsulation of certain anesthetics and tomodulate the release rate of the encapsulated anesthetics. The inventionalso provides methods of making such compositions, and of providinglocal anesthesia to subjects by administering the compositions.

The prior art has used commonly available monoprotic (for example,hydrochloride or glutamic) salts of biologically active compounds. Thishas resulted in either unacceptable formulations for encapsulating thebiologically active substances in MVL or very low encapsulationefficiency. The invention results from the surprising finding thatinclusion of the free base form of anesthetic compounds solublized withphosphoric acid, or conversion of the commonly available hydrochloridesalts of anesthetic compounds into phosphate (salt of triprotic mineralacid) or sulfate salts (salt of diprotic mineral acid) for inclusioninto MVL results in markedly improved encapsulation efficiency as wellas sustained release in biologically relevant media. Polyalcoholicorganic acids such as glucuronic or gluconic acid are also included,wherein such acid is co-encapsulated with anesthetics to assistencapsulation and to effect sustained-release of the anesthetic.Surprisingly, polyalcoholic organic acids are superior tonon-polyalcoholic organic acids, giving compositions with highencapsulation efficiency and sustained release of anesthetic.Polyalcoholic organic acids greatly improve the encapsulation ofanesthetic and the acceptability of the formulation. Sulfate salts and anumber of other salts require the inclusion of such acids to formacceptable formulations.

When the encapsulated anesthetic is administered as a singleintracutaneous or subcutaneous dose, the duration of anesthesia andhalf-life of the drug at the local injection site is increased ascompared to injection of unencapsulated anesthetic. The maximumtolerated dose of the encapsulated anesthetic is also markedly increasedin the liposomal formulation over injection of unencapsulatedanesthetic.

The major use for the invention is for making sustained-releaseformulations of biologically active substances that have high diffusionrates through bilayer lipid membranes. Both the use of diprotic andtriprotic mineral acid salts of biologically active substances andcon-encapsulation of polyalcoholic organic acids enable thesedifficult-to-encapsulate drugs to be encapsulated easily and releasedslowly.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing the anesthetic effect (number of nonresponses to six pin-pricks) as a function of time following a singleintracutaneous dose of MVL-encapsulated bupivacaine phosphate containingdifferent concentrations of bupivacaine.

FIG. 1B is a graph showing the anesthetic effect (number of nonresponses to six pin-pricks) as a function of time following a singleintracutaneous dose of unencapsulated bupivacaine hydrochloride atdifferent concentrations.

FIG. 2 is a graph showing a comparison of the duration of anesthesia forthe formulations of FIGS. 1A (MVL-encapsulated bupivacaine phosphate,filled circles) and 1B (unencapsulated bupivacaine hydrochloride, opencircles) as quantified by “time to half maximal response (R₃)”(ordinate) versus concentration of administered dose (abscissa).

FIG. 3A is a graph showing the total amount of bupivacaine (mg)remaining at an injection site up to 72 hours following a singleintracutaneous dose of MVL-encapsulated bupivacaine phosphate (filledcircles) or unencapsulated bupivacaine hydrochloride (open circles).

FIG. 3B is a graph showing the serum bupivacaine concentrations (μg/mL)up to 72 hours following a single intracutaneous dose ofMVL-encapsulated bupivacaine phosphate at a concentration of 1.0 percent(w/v) of bupivacaine (filled circles) or 0.5 percent (w/v) ofunencapsulated bupivacaine hydrochloride (open circles).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Post-operative or post-trauma pain is thought to be most intense in theimmediate post-operative or post-injury and subsequent 24-hour period.It is possible that improved control of post-operative pain can decreasepulmonary and gastrointestinal complications and perhaps shortenhospital stay. Systemic opiates commonly used to control pain duringthis post-operative period can depress pulmonary function and slowgastro-intestinal recovery. Other antinociceptive agents such asnon-steroidal anti-inflammatory agent ketorolac tromethamine canincrease bleeding and gastrointestinal irritation in this time ofstress. Since nociceptive stimuli arising from surgical interventions ortraumatic injury are usually local or regional in origin, prolongedlocal or regional sensory block for pain control is an intriguingconcept. Thus, it is believed that improved treatment with localanesthetics involves maintenance of anesthetic level for a prolongedperiod. Unfortunately, the half-life of many anesthetics is very shortafter an intraperitoneal (IP), intravenous (IV), intrathecal (IT),intraartricular (IA), intramuscular (IM), or subcutaneous (SC) dose.Therefore, a slow-release preparation which provides a prolonged andsustained exposure at a therapeutic concentration of a local anestheticis needed. The present invention is directed to the production,composition, and use of such a preparation.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar to those described herein can be used in the practice or testingof the present invention, suitable methods and materials are describedbelow. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

Anesthetics

The present invention provides prolonged release of local anesthetics,particularly of the “amide-type” anesthetics, from MVL followingadministration of compositions containing the MVL. The inventionutilizes a local anesthetic encapsulated in MVL. The local anestheticgenerally belongs to the class known as the amide-type anesthetics. Thename comes from the presence of the amide (—NHCO—) linkage in thecentral portion of the molecule. The group linked to the nitrogen end ofthe amide is a substituted phenyl ring, especially a phenyl ringcontaining at least one short chain alkyl group, such as methyl, ethyl,propyl or butyl. Examples of such groups include 2-methylphenyl,2,6-dimethylphenyl, 2-ethylphenyl, 2,6-diethylphenyl, and2-ethyl-6-methylphenyl. If the substituent group is 2,6-dimethylphenyl,the local anesthetics are also referred to as 2,6-xylidide anesthetics.

The group linked to the CO end of the amide linkage is designated asCHR₁R₂. In the foregoing designation, R₁ is a secondary or tertiaryalkyl amine such as N-alkyl amine or N,N-dialkyl amine. Short chainalkyl groups (from one to four carbon atoms) are preferred. Examplesinclude N-methylamine, N-ethylamine, N-propylamine, N-butylamine,N,N-dimethylamine, N,N-diethylamine, N-ethyl-N-methylamine, andsimilarly constructed substituents. The three and four member alkylchains can be of any configuration, that is, straight chain (n-alkyl),or branched (iso-, sec-, or tert-alkyl). Alternatively, R₁ can be asecondary or tertiary alkyleneamino group, which further links to R₂.For example, R₁ and R₂ can be linked by a secondary or tertiarynitrogen-containing alkyl chain, to form an N-alkyl substitutedpiperidine or pyrrolidine ring. In such examples, the N-alkyl group ispreferably a short chain (one to four carbon atoms), such as N-methyl,N-ethyl, N-propyl or N-butyl, in which the chain can be straight orbranched. The R₁-R₂ linking substituent can be 2-piperidyl,2-pyrrolidyl, 3-piperidyl, 3-pyrrolidyl, 4-piperidyl or 4-pyrrolidyl.Preferably, the substituent formed when R₁ and R₂ are linked by asecondary or tertiary nitrogen-containing alkylene chain is 2-piperidylor 2-pyrrolidyl. The stereochemistry of the compounds can be either R orS, depending on the most efficient anesthetic activity. For example,commercially available ropivacaine is found in the (S)(−) configuration.Bupivacaine is also found in the form known as levo-bupivacaine. In theforegoing designation, R₂ is either hydrogen, short chain alkyl (one tofour carbon atoms) or a secondary or tertiary alkyleneamino chain whichlinks to R₁, as described above.

The amide-type anesthetics which are useful in the present invention aredescribed by the following structure:

wherein R₁, and R₂ are as described above, and R₃ is analkyl-substituted phenyl ring, as described above.

Illustrative of the forgoing description of the amide-type anestheticsuseful in the present invention are, for example, bupivacaine,levo-bupivacaine, mepivacaine, lidocaine, pyrrocaine, prilocaine, andropivacaine.

The anesthetics should be present in the compositions of the inventionin concentrations of from about 0.01% to about 5.0% w/v, or preferablyfrom about 0.5% to about 2.0% w/v. The weight percentages are defined asweight of anesthetic per volume of MVL.

The free base forms of local anesthetics of the invention can besolublized. Desirably, the water-soluble salt form is formed for theirstorage and delivery from MVL. The salt form can be introduced to thefirst aqueous phase of the MVL as such, or can be formed by adding thefree base form, and sufficient acid to solublize the anesthetics to thedesired extent. The salt can be any pharmaceutically acceptable di- ortri-protic mineral salt, such as the phosphate or sulfate salt. Alsouseful are the polyhydroxyl carboxylic acid salts of the anesthetic,such as the tartarate, gluconate or gluconurate salts. Combinations ofsuch salts are preferable as components of the first aqueous phase ofthe inventive compositions. Thus, amide-type anesthetics are present inthe pharmaceutical compositions of the invention in the form ofpolyhydroxy carboxylate salts, and di- and tri-protic mineral salts.Preferred embodiments of the invention are those with a binary mixtureof amide-type anesthetic salts, one derived from a polyhydroxycarboxylic acid, and the other derived from a di- or tri-protic mineralacid.

Multivesicular Liposomes

The anesthetic compositions of the invention also include multivesicularliposomes (MVL) which encapsulate and provide modulated and sustainedrelease of the anesthetics described above. The MVL are made by thefollowing process. A “water-in-oil” type emulsion containing anon-hydrohalic acid salt of any of the anesthetics described above isformed from two immiscible phases, a lipid phase and a first aqueousphase.

The lipid phase is made up of at least one amphipathic lipid and atleast one neutral lipid in a volatile organic solvent. The term“amphipathic lipid” refers to molecules having a hydrophilic “head”group and a hydrophobic “tail” group and may have membrane-formingcapability. As used herein, amphipathic lipids include those having anet negative charge, a net positive charge, and zwitterionic lipids(having no net charge at their isoelectric point). The term “neutrallipid” refers to oils or fats that have no vesicle-forming capability bythemselves, and lack a charged or hydrophilic “head” group. Examples ofneutral lipids include, but are not limited to, glycerol esters, glycolesters, tocopherol esters, sterol esters which lack a charged orhydrophilic “head” group, and alkanes and squalenes.

The amphipathic lipid is chosen from a wide range of lipids having ahydrophobic region and a hydrophilic region in the same molecule.Suitable amphipathic lipids are zwitterionic phospholipids, includingphosphatidylcholine, phosphatidylethanolamines, sphingomyelins,lysophosphatidylcholines, and lysophosphatidylethanolamines. Alsosuitable are the anionic amphipathic phospholipids such asphosphatidylglycerols, phosphatidylserines, phosphatidylinositols,phosphatidic acids, and cardiolipins. Also suitable are the cationicamphipathic lipids such as acyl trimethylammonium propanes, diacyldimethylammonium propanes, and stearylamine.

Suitable neutral lipids are triglycerides, propylene glycol esters,ethylene glycol esters, and squalene. Examples of triglycerides usefulin the present invention are triolein, tripalmitolein, trimyristolein,trilinolein, tributyrin, tricaproin, tricaprylin, and tricaprin. Thefatty chains in the triglycerides useful in the present invention can beall the same, or not all the same (mixed chain triglycerides), includingall different. Both saturated and unsaturated fatty chains are useful inthe present invention. The propylene glycol esters can be mixed diestersof caprylic and capric acids.

Many types of volatile organic solvents can be used in the presentinvention, including ethers, esters, halogenated ethers, hydrocarbons,halohydrocarbons, or Freons. For example, diethyl ether, chloroform,tetrahydrofuran, ethyl acetate, Forane, and any combinations thereof aresuitable for use in making the anesthetic compositions of the presentinvention.

Optionally, but highly desirably, other components are included in thelipid phase. Among these are cholesterol or plant sterols.

The first aqueous phase includes an anesthetic, at least one polyhydroxycarboxylic acid, and at least one di- or tri-protic mineral acid. Insome embodiments of the invention, also included is hydrochloric acid.Hydrochloric acid is not an essential constituent, but rather isoptional and desirable in some embodiments. The di- or tri-proticmineral acids include sulfuric acid, and phosphoric acid. Also includedin the first aqueous phase are such polyhydroxy carboxylic acids asglucuronic acid, gluconic acid, and tartaric acid. The di- andtri-protic mineral acids and the polyhydroxy organic acids are presentin the first aqueous phase in concentrations of from 0.01 mM to about0.5 M, or preferably from about 5 mM to about 300 mM. When hydrochloricacid is used, it is present in lower amounts, from about 0.1 mM to about50 mM, or preferably from about 0.5 mM to about 25 mM.

The lipid phase and first aqueous phase are mixed by mechanicalturbulence, such as through use of rotating or vibrating blades,shaking, extrusion through baffled structures or porous pipes, byultrasound, or by nozzle atomization, to produce a water-in-oilemulsion. Thus, the anesthetics of the invention are encapsulateddirectly in the first step of MVL manufacture.

The whole water-in-oil emulsion is then dispersed into a second aqueousphase by means described above, to form solvent spherules suspended inthe second aqueous phase. The term “solvent spherules” refers to amicroscopic spheroid droplet of organic solvent, within which aresuspended multiple smaller droplets of aqueous solution. The resultingsolvent spherules therefore contain multiple aqueous droplets with theanesthetic dissolved therein. The second aqueous phase can containadditional components such as glucose, and/or lysine.

The volatile organic solvent is then removed from the spherules, forinstance by surface evaporation from the suspension: When the solvent issubstantially or completely evaporated, MVL are formed. Gases which canbe used for the evaporation include nitrogen, argon, helium, oxygen,hydrogen, and carbon dioxide. Alternatively, the volatile solvent can beremoved by sparging, rotary evaporation, or with the use of solventselective membranes.

Method of Providing Anesthesia

The invention also provides a method of providing regional aesthesia toa subject by administering the claimed anesthetic compositions eitherintracutaneously, subcutaneously or via a local or regional nerve block.The dosages can be administered either as a nerve block (including tothe limit of acting as a motor block), or as a sensory block.

The term “therapeutically effective” as it pertains to the compositionsof this invention means that an anesthetic present in the first aqueousphase within the MVL is released in a manner sufficient to achieve aparticular level of anesthesia. Exact dosages will vary depending onsuch factors as the particular anesthetic, as well as patient factorssuch as age, sex, general condition, and the like. Those of skill in theart can readily take these factors into account and use them toestablish effective therapeutic concentrations without resort to undueexperimentation.

Generally however, the dosage range appropriate for human use includesthe range of from about 20 mg to about 300 mg of total anesthetic. Theupper limit is limited by the toxicity of the particular anesthetic, andthe lower limit is approximately 10% of the upper limit.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES

The following examples illustrate the preparation and properties ofcertain embodiments of the present invention.

Example 1 Manufacture of Bupivacaine-Phosphate-Containing MVL

Bupivacaine hydrochloride (Sigma Chemical Co., St. Louis, Mo.) wasconverted into bupivacaine phosphate by initial precipitation of aqueousbupivacaine hydrochloride with 1N sodium hydroxide to prepare the freebase. The precipitate was extensively washed with water, and thenconverted into phosphate salt with an equimolar amount of phosphoricacid.

For each batch of the formulation, 5 mL of a discontinuous first aqueouscomponent containing 60 mg/mL of bupivacaine phosphate, 150 mMglucuronic acid, 15 mN hydrochloric acid, and 20 mM phosphoric acid wasadded to a mixer vessel containing a lipid component containing 5 mL ofUSP chloroform (Spectrum Chemical Co., Gardena, Calif.) as solvent, and18.6 mM 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 4.2 mMdipalmitoyl phosphatidylglycerol (Avanti Polar-Lipids, Inc., Alabaster,Ala.) (an anionic ampbipathic lipid), 30 mM cholesterol (Avanti Lipids),and 10.8 mM tricaprylin. The immiscible first aqueous component andlipid component were mixed at 16,000 rpm in an Omni-mixer (OMNIInternational, Gainesville, Va.) for 9 minutes. The resultingwater-in-oil emulsion was transferred to a 50 mL mixing vesselcontaining 25 mL of a continuous second aqueous component containing 32mg/mL of glucose and 10 mM free-base lysine (Sigma Chemical Co., St.Louis, Mo.). The mixture was then mixed for 20 seconds at 4000 rpm in anOmni mixer.

The resulting water-in-oil-in-water double emulsion was transferred to a1 L Erlenmeyer flask containing 275 mL of the continuous second aqueousphase (glucose, 32 mg/mL; free-base lysine, 40 mM). The chloroform wasevaporated for 15 minutes under a constant flow (90 L/min) of nitrogengas at 37° C. to form MVL particles in suspension. The MVL particleswere isolated by centrifugation at 200×g for 10 minutes, then washedthree times with a 0.9 percent (w/v) solution of NaCl. Each batch wasstored at 2-8° C. and used for subsequent studies within 48 hours.

Example 2 Recovery of Bupivacaine from Different MVL Formulations

The bupivacaine samples were solublized by adding an equimolar volume ofa 1 M solution of the indicated acid and then slowly adding, withstirring, additional water until 60 mg/mL or a clear solution wasachieved. The pH was then adjusted to approximately 5. The finalbupivacaine concentration was determined by HPLC against an internalstandard.

For each formulation attempt, the first aqueous phase solution containedthe bupivacaine counterion at 60 mg bupivacaine per mL, or the limit ofsolubility of the bupivacaine counterion, at pH 5. Other parameters forMVL manufacture was as described above. Recovery refers to percent ofbupivacaine in counterion solution encapsulated and recovered in finalMVL product. For study 2, the first aqueous phase also contained 150 mMglucuronic acid. The results are shown in Table 1.

TABLE 1 Recovery of MVL-encapsulated bupivacaine from FormulationsContaining Various Acids Bupivacaine (mg/mL) acid included additionalacid recovery (%) Study 1 60 phosphoric —  5.7 60 sulfuric — (clumped)23 nitric — no MVL formed 40 hydrochloric — no MVL formed 26 glucuronic— 34 60 tartaric — clumped 41 acetic — no MVL formed 2.2 perchloric — noMVL formed Study 2 60 phosphoric 150 mM glucuronic 35 60 sulfuric 150 mMglucuronic 16 23 nitric 150 mM glucuronic 45 40 hydrochloric 150 mMglucuronic 16 26 glucuronic 150 mM glucuronic 48 60 tartaric 150 mMglucuronic 20 41 acetic 150 mM glucuronic 18 2.2 perchloric 150 mMglucuronic no MVL formed 60 citric 150 mM glucuronic 13 60 malic 150 mMglucuronic 19 60 succinic 150 mM glucuronic 20

The results in Table 1 demonstrate clearly that the addition of apolyhydroxy organic acid (in this case, glucuronic acid) in addition toone of a number of other acids, including triprotic mineral acids suchas phosphoric acid, or polyhydroxy organic acids such as glucuronic acidprovides a remarkable synergistic effect. This surprising discoveryleads to higher loading and recovery of the MVL of the invention thanpreviously found.

Example 3 In Vivo Animal Studies Using Intracutaneous Injections

Male guinea pigs weighing 800-1000 grams (Harlan-Sprague-Dawley, SanDiego, Calif.) were used for efficacy studies. Male guinea pigs(Harlan-Sprague-Dawley) weighing 400-600 grams were used forpharmacokinetic studies. The animals were housed, 1 per cage, in atemperature-controlled environment with alternating 12-hour periods oflight and darkness and given unrestricted access to food and water.Prior to each study, animals were habituated to the environment for atleast 7 days. Female CDI mice (Sprague-Dawley) weighing 22-28 grams wereused for determination of maximum tolerated dose (MTD). All animals weremaintained in accordance with guidelines of the Committee on Care andUse of Laboratory Animals of the Institute of Laboratory AnimalResources, National Research Council.

The formulations of MVL-encapsulated bupivacaine and bupivacainehydrochloride prepared as described above were diluted in normal salineso that a constant volume of 1 mL contained a dose at concentrations of2.1%, 1.0%, or 0.5% (w/v) bupivacaine. Concentrations were confirmed bysolubilizing a 50 μl aliquot of the MVL formulation in 1 mL of isopropylalcohol followed by dilution in water and assay by a previouslypublished HPLC method as described (P. Le Guevello et al., JChromatography 622:284-290, 1993). The HPLC analysis of the MVLformulations revealed that less than 5% of total bupivacaine was presentin the formulation as unencapsulated bupivacaine.

Infiltration anesthesia studies were performed in the test guinea pigsusing a modified intracutaneous wheal pin-prick model as described (R.H. de Jong et al., Anesth. Analog 59:401-5, 1980). On the day precedingthe experiment, hairs on the backs of the animals were clipped. Eachanimal received either a dose of MVL-encapsulated bupivacaine(concentrations of 0.5, 1.0 or 2.1 percent (w/v) bupivacaine) orunencapsulated bupivacaine (concentrations of 0.25, 0.5, 0.75 or 1.0(w/v) percent bupivacaine), which created a wheal. The margin of thewheal was marked with indelible ink. The reaction to pin pricks at thesite of injection was tested just prior to injection (time zero) and 15minutes, 3, 6, 12, 18, 24, 30 and 36 hours following injection ofMVL-encapsulated bupivacaine, and zero, 5, 15, minutes, 1, 1.5, 2, 3, 4,5, 6, 7 and 8 hours following injection of bupivacaine hydrochloride.The pin pricks were applied first to a control area outside the wheal ateach time point. After observing the animal's normal reaction to the pinprick (vocalization response), six pricks were applied inside the whealand the number of pricks to which the guinea-pig failed to react wererecorded as nonresponses. Each prick was applied at an interval of 3-5sec. All animals responded with vocalization to all six pin pricks atbaseline.

The animal data obtained indicate a rapid onset of anesthesia followinga single intracutaneous dose of bupivacaine encapsulated in MVL,followed by a prolonged duration of sensory anesthesia lasting up to 28hours, depending on the concentration of bupivacaine in the MVLadministered. The rapid onset of anesthesia is attributable, in part, toa low, but a significant fraction of unencapsulated bupivacaine(approximately 5% of the total) in the batches of MVL encapsulatingbupivacaine used in these experiments. The duration of anesthesiaobtained by use of these formulations may cover the worst post-operativeperiod, the first 24 hours. A longer anesthesia duration, perhaps 7 daysor longer, would be more suitable for chronic pain, such as cancer orneuropathic pain.

Example 4 Data Analysis of Efficacy Studies

Anesthetic efficacy curves were plotted as the number of nonresponses asa function of time. Areas under the curve (AUC) were calculated by thetrapezoidal rule to the last data point. With regard to FIG. 1A, theMVL-encapsulated bupivacaine concentrations by weight per volume percent(w/v %) were 2.1% (●), 1.0 (▪), and 0.5% (▴). With regard to FIG. 1B,unencapsulated bupivacaine concentrations by weight per volume percentwere 0.25% (∇), 0.5% (Δ), 0.75% (◯), and 1.0% (□). Each data pointrepresents the average for 5 to 6 animals. The error bars represent thestandard error of mean (SEM).

Evaluation of response to pin pricks showed that complete localanesthesia (no response condition) was achieved within 15 minutesfollowing intracutaneous administration of either the MVL formulation ofbupivacaine (FIG. 1A) or of unencapsulated bupivacaine hydrochloride(FIG. 1B).

FIG. 2 shows the duration of anesthetic effect as measured by the timeto half maximal response (R₃) for the various doses of the MVLformulation (filled circles) and for unencapsulated drug (open circles).Each data point represents the average and standard error of mean (SEM)from 5 to 6 animals. These results show that the duration of anestheticeffect was concentration dependent in both cases. However, the MVLformulations containing concentrations 0.5 and 1.0 percent by weight ofbupivacaine phosphate were prolonged 3.2 and 2.9-fold, respectively, ascompared to comparable doses of bupivacaine hydrochloride.

Example 5 Determination of Maximum Tolerated Dose (MTD)

Determination of maximum tolerated dose (MTD) was done in mice using asubcutaneous test known in the art (R. H. de long et al., Anesthesiology54:177-81, 1981). Groups of three mice each were given injections ofeither 780 or 980 mg per kg body weight of the above-described MVLformulation containing bupivacaine sulfate as two divided doses of 500μl each (total 1.0 mL volume). The doses were administered in rapidsequence into each flank. Control groups of 3 mice each received one ofthe test doses as a single dose of 10, 20, 30 or 50 mg/kg body weight ofunencapsulated bupivacaine hydrochloride. MTD was defined as the highestdose at which none of the animals experienced systemic toxicity.

These studies showed that none of the mice that received freebupivacaine hydrochloride subcutaneously showed any signs of systemictoxicity at doses of 10 and 20 mg/kg. However, at 30 and 50 mg/kg doses,two out of three, and three out of three animals, respectively,developed toxicity. On the other hand, the MVL formulation ofbupivacaine sulfate administered subcutaneously at a dose of 780 mg/kgshowed no sign of systemic toxicity in any of the animals; whereas threeout of three animals had toxicity at a dose of 980 mg/kg. Therefore, theMTD for unencapsulated bupivacaine hydrochloride was estimated to beabout 20 mg/kg of body weight, and that for MVL-encapsulated bupivacainesulfate was estimated at about 780 mg/kg of body weight.

The most serious toxicity arising from the use of local anesthetics isseizure or cardiovascular collapse. Consistent with the lower peak serumconcentration found following administration of the MVL formulations ofbupivacaine, the maximum tolerated dose for the MVL-encapsulatedbupivacaine was many times higher than that for bupivacainehydrochloride. These data would predict an increased systemic safetyprofile for the compositions produced by the method of this invention.The toxicity profiles of the active and inactive ingredients arewell-defined, reducing the likelihood of finding unexpected toxicity.

Example 6 Pharmacokinetic Studies

The in vivo pharmacokinetics of the MVL formulations of bupivacaine andfree bupivacaine hydrochloride were compared following a single 1 mLintracutaneous dose of the MVL formulation containing 1.0 percent (w/v)of bupivacaine, or a dose of 0.5 percent (w/v) of unencapsulatedbupivacaine hydrochloride to a group of guinea pigs. The lowerconcentration was selected for bupivacaine hydrochloride because the400-600 gram animals were unable to tolerate a 1.0% concentration doseof the unencapsulated drug. For the animals that received freebupivacaine hydrochloride, samples were collected at 0 and 30 minutes,and 1, 3, 6 and 9 hours following injection, while the animals thatreceived the MVL formulations of bupivacaine were sampled 0, 6, 12, 18,24, 48, and 72 hours following injection. At each time point, 3 or moreanimals were first anesthetized with halothane and then exanguinated bycardiac puncture. Serum samples were obtained by centrifugation ofclotted whole blood. Skin was collected around the injection site with 3cm margins, together with a 2-3 mm layer of underlying subcutaneoustissue. The skin and serum samples were kept frozen at −20° C. untilanalysis.

The amount of total bupivacaine remaining in the injection site wasobtained by mincing the tissue followed by in toto homogenization inwater using a Polytron homogenizer (Brinkman, Littau, Switzerland).Bupivacaine was extracted from the homogenate and analyzed by HPLC usinga previously published method (Le Guevello et al., J. Chromatography622:284-290, 1993). Bupivacaine concentration in serum was determined byextraction followed by HPLC (Le Guevello et al., supra). Tetracainespiked into each sample before extraction was used as an internalstandard. The limit of detection of the assay was 20 ng/mL.

Pharmacokinetic data obtained from the samples were analyzed using anoncompartmental model (WinNonlin software, Scientific Consulting, Inc.,Apex, N.C.). Parameters calculated were the drug amount remaining at theinjection site, the area under the “amount vs. time” curve (AUC), anddrug half-life (t_(1/2,)). In addition to the AUC and half-life, peakconcentration (C_(max)) was also reported for serum bupivacainepharmacokinetics.

One way analysis of variance (ANOVA) was used to separately determinedose dependency for the different drug formulations and route ofadministration (via MVL or free drug) as well as for comparison betweenformulations. Student-Newman-Keuls tests were used on all ANOVAanalyses. The pharmacokinetic parameters obtained by these methods aresummarized in Table 2 below.

TABLE 2 Pharmacokinetic Parameters Following Either a Single 1.0%DepoBupivacaine or 0.5% Bupivacaine Hydrochloride IntracutaneousInjection MVL-encapsulated Bupivacaine Bupivacaine hydrochloride DrugConcentration Administered 1.0% 0.5% Peak Amount (mg), Skin 11.6 3.8t_(1/2) (hr), Skin 12.0 1.3 AUC (mg * hr), Skin 236 2.9 C_(max) (μg/mL),Serum 2.9 6.5 t_(1/2) (hr), Serum 20.5 2.1 AUC (μg * hr * mL⁻¹), Serum56.1 21.2 r², Skin 0.97 0.85 r², Serum 0.89 0.93

The “drug concentration administered” is in units of weight ofanesthetic per volume of MVL. The “peak amount” shows the maximum amountof the indicated substance in the skin sample. The “C_(max)” is themaximum concentration of the indicated substance in serum. The “t_(1/2)”is the drug half-life. The “AUC area” is the area under the “amount vs.time” curve. The “r²” is the square of the sample correlationcoefficient.

As these results show, following intracutaneous administration of theMVL formulation, the total amount of drug in the injection-site tissuedecreased with a half-life of 12 hours compared to 1.3 hours forunencapsulated bupivacaine hydrochloride. Peak serum concentration ofbupivacaine following a single intracutaneous dose of the 1.0% MVLformulation was decreased 2.2 fold (4.4 fold when corrected for thedose) compared to that following 0.5% bupivacaine hydrochloride.Similarly, the terminal serum half-life for 1.0 percent (w/v) MVLformulations was 20.5 hours compared to 2.1 hours for unencapsulatedbupivacaine hydrochloride at a concentration of 0.5 percent (w/v).

The local injection site AUC for the MVL formulation was 81 times (41times when corrected for the dose) that for unencapsulated bupivacainehydrochloride, and the serum AUC was 2.6 times (1.3 times when correctedfor the dose) that for bupivacaine hydrochloride.

FIGS. 3A and 3B show the result of the pharmacokinetic studies. FIG. 3Ashows the amount of either MVL-encapsulated bupivacaine at aconcentration of 1.0 percent (w/v) of bupivacaine (filled circles) orunencapsulated bupivacaine hydrochloride at a concentration of 0.5percent (w/v) of bupivacaine (open circles) remaining at an injectionsite at time points tested over a period of 72 hours. FIG. 3B shows theserum concentration (μg/mL) of bupivacaine following a singleintracutaneous dose of the MVL-encapsulated formulation at 1.0 percent(w/v) of bupivacaine (filled circles) or unencapsulated bupivacainehydrochloride at a concentration of 0.5 percent (w/v) of bupivacaine(open circles). Each data point represents the average and standarderror of mean (SEM) from 3 to 6 animals. A statistical significancelevel of 0.05 was used for all tests.

The pharmacokinetics data obtained in the Examples herein wereconsistent with a prolonged duration of anesthetic effect. Theanesthesia duration was 2.9- to 3.2-fold longer for bupivacaineencapsulated in MVL, and the half-life at the injection site was9.2-fold longer compared to bupivacaine hydrochloride. The peak serumconcentration was decreased by 4.5 fold (normalized to equivalentdoses), and the terminal serum half-life was increased by 9.8-foldfollowing administration of bupivacaine encapsulated in MVL compared tobupivacaine hydrochloride.

In conclusion, a single intracutaneous dose of bupivacaine encapsulatedin MVL resulted in a prolonged duration of anesthesia (up to 28 hours)and a 9.2-fold (uncorrected for dose) increase in local injection-sitehalf-life compared to bupivacaine hydrochloride. The maximum tolerateddose was increased by 39 fold compared to bupivacaine hydrochloride.Therefore the formulations of the invention have utility for sustainedinfiltration anesthesia without the need for continuous infusion and mayincrease patient satisfaction.

A number of embodiments of the present invention have been described.Nevertheless, it will be understood that various aspects, advantages andmodifications of the invention may be made without departing from thespirit and scope of the invention. The foregoing description is intendedto illustrate and not limit the scope of the invention, which is definedby the scope of the appended claims.

What is claimed is:
 1. A process for making a multivesicular liposomeformulation comprising bupivacaine phosphate comprising: a) preparing afirst aqueous component comprising at least one acid in sufficientquantity to solubilize bupivacaine or a salt thereof, said acid beingphosphoric acid; b) preparing a lipid component comprising at least oneorganic solvent, at least one amphipathic lipid, and at least oneneutral lipid lacking a hydrophilic head group; c) mixing said firstaqueous component and said lipid component to form a water-in-oilemulsion, wherein at least one component comprises bupivacaine or a saltthereof; d) mixing said water-in-oil emulsion with a second aqueouscomponent to form solvent spherules; and e) removing the organic solventfrom the solvent spherules to form multivesicular liposomes.
 2. Theprocess of claim 1, wherein said amphipathic lipid isdierucoylphosphatidyl choline.
 3. The process of claim 1, wherein saidamphipathic lipid is dipalmitoylphosphatidylglycerol.
 4. The process ofclaim 1, wherein said neutral lipid is tricaprylin.
 5. A pharmaceuticalcomposition comprising multivesicular liposomes, said multivesicularliposome comprising: bupivacaine phosphate; a lipid component comprisingat least one amphipathic lipid and at least one neutral lipid lacking ahydrophilic head group; and, optionally, a cholesterol and/or a plantsterol.
 6. The pharmaceutical composition of claim 5, wherein said atleast one amphipathic lipid is selected from the group consisting ofdierucoylphosphatidylcholine and dipalmitoylphosphatidylglycerol.
 7. Thepharmaceutical composition of claim 5, wherein said at least oneamphipathic lipid comprises dierucoylphosphatidylcholine.
 8. Thepharmaceutical composition of claim 5, wherein said at least oneamphipathic lipid comprises dipalmitoylphosphatidylglycerol.
 9. Thepharmaceutical composition of claim 5, wherein said at least one neutrallipid is tricaprylin.
 10. The pharmaceutical composition of claim 5,wherein said at least one amphipathic lipid comprisesdierucoylphosphatidylcholine and/or dipalmitoylphosphatidylglycerol, andsaid at least one neutral lipid comprises tricaprylin.
 11. Thepharmaceutical composition of claim 5, wherein the amphipathic lipid isdierucoylphosphatidylcholine and dipalmitoylphosphatidylglycerol; andeach neutral lipid is selected from the group consisting of triolein,tripalmitolein, trimyristolein, trilinolein, tributyrin, tricaproin,tricaprylin, and tricaprin.
 12. The pharmaceutical composition of claim11, wherein the neutral lipid is tricaprylin.
 13. The pharmaceuticalcomposition of claim 12, including cholesterol.
 14. The pharmaceuticalcomposition of claim 5, wherein the composition includes from about0.01% w/v to about 5.0% w/v of bupivacaine phosphate.
 15. A methodproviding regional anesthesia to a subject in need thereof byadministering in a single dose a composition of claim
 5. 16. The methodof claim 15, wherein the single dose includes from about 20 mg to about300 mg of bupivacaine phosphate.
 17. The method of claim 15, wherein thesingle dose administration is intracutaneously, subcutaneously, or vianerve block.
 18. The method of claim 17, wherein the nerve block islocal or regional nerve block.
 19. The method of claim 15, wherein theregional anesthesia has a duration of 12 hours or more.
 20. A method oftreating post-operative or post-trauma pain in a subject in need thereofcomprising administering in a single dose a composition of claim
 5. 21.The method of claim 20, wherein said at least one amphipathic lipidcomprises dierucoylphosphatidylcholine.
 22. The method of claim 20,wherein said at least one neutral lipid is tricaprylin.
 23. The methodof claim 20, wherein the single dose administration provides pain reliefof 12 hours or more.